1. Field of the Invention
This invention relates to a highly specific enzyme for hydrolysis of complex polysaccharides. More specifically, there is disclosed herein novel compositions comprising, feraxanase, an enzyme capable of specifically degrading feraxan, a feruloylated arabinoxylan found in plant cell walls.
This invention was made with Government support under Grant No. DMB 85-05901 with the National Science Foundation and the University of California. The Government has certain rights in this invention.
2. Information Disclosure
The compositions of this invention selectively cleave feraxan from arabinoxylans commonly found in plant cell walls. Arabinoxylans are major components of plant cell walls, in particular cereal plant cell walls. See for example, Carpita, N.C., "Hemicellulosic Polymers of Cell Walls of Zea Coleoptiles," Plant Physiol. 72:515-521 (1983); Dubois et al., "Colorimetric Method for Determination of Sugars and Related Substances," Anal. Chem. 28:350-356 (1956); and Wilkie, "The Hemicelluloses of Grasses and Cereals," Adv. Carbohydr. Chem. Biochem. 36:215-264 (1979). Structural analyses have been conducted on arabinoxylans in an attempt to elucidate the chemical basis underlying alterations in the physical properties of the cell wall responsible for growth and development of the plant tissues. Carpita, "Cell wall Development in Maize Coleoptiles," Plant Physiol. 76:205-212 (1984); Carpita, "Incorporation of Proline and Aromatic Amino Acids into Cell Walls of Maize Coleoptiles," Plant Physiol. 80:660-666 (1986); Darvill et al., "Structure of Plant Cell Walls, XI Glucuronoarabinoxylan, a Second Hemicellulose in the Primary Cell Walls of Suspension-Cultured Sycamore Cells," Plant Physiol. 66:1135-1139 (1980); Yamamoto et al., "Effects of Auxin on the Structure of Hemicelluloses of Avena Coleoptiles," Plant Cell Physiol. 21:373-381 (1980).
Although recent studies have shown that phenolic components, particularly ferulic acid, are attached to arabinoxylans, the structure of the feruloylated-arabinoxylans (feraxan) of the cell wall has not been elucidated. In most structural studies of feraxans, alkaline solutions have been used to extract matrix polysaccharides. See, for example, Carpita, Plant Physiol. (1983); Darvill et al., Plant Physiol. (1980); Yamamoto et al.; Wilkie, all supra. Alkaline extraction, however, results in not only nonselective liberation of matrix polysaccharides but in undersirable cleavage of certain covalent bonds, particularly ester linkages through which phenolic acids are attached to arabinoxylans.
To avoid the problems encountered in the alkaline extraction procedures, selective dissociations of cell wall fragments by purified enzymes with known specificities have been used. Most success thus far has been reported for the pectic polysaccharides. See, Talmadge et al., "The Structure of Plant Cell Walls I.," Plant Physiol. 51:158-173 (1973). Success has also been reported with (1.fwdarw.3), (1.fwdarw.4)-.beta.-D-glucans. Huber and Nevins, "Preparation and Properties of a .beta.-D-glucanase for the Specific Hydrolysis of .beta.-D-glucans." Plant Physiol. 601:300-304 (1977).
In order to better understand the role of arabinoxylans in the overall scheme of plant cell wall structure, enzymes having selective sites of polymer cleavage are needed. At present, Kato and Nevins have purified a (1.fwdarw.4)-.beta.-D-xylanase capable of degrading feraxan of maize cell wall from a B. subtilis enzyme preparation. This enzyme, however, had limited action in dissociating feraxan from the maize cell wall. Only about 10% of feraxan present in the wall was released. Kato and Nevins, "Enzymic Dissociation of Zea Shoot Cell Wall Polysaccharides, III," Plant Physiol. 75:753-758 (1984). Thus, there is a need for additional enzymes for use in cell wall structural analysis. In addition there is a need for selective enzymes for use in biomass conversion of plant material from waste into animal feed as undigestible carbohydrates are enzymatically converted to digestible sugars. Novel enzyme systems are also needed for production of specific sugars from polymers. Finally, there is a growing need for selective enzymes able to partially digest cell walls of plants to facilitate genetic transformations using recombinant genetics.